Method for enhancing the dna repair process, treating disorders associated with the dna repair process, enhancing antitumor response and treating disorders associated with anti-tumor response in mammals by administering purified quinic acid and/or carboxy alkyl ester

ABSTRACT

For use in mammals, methods for enhancing the anti-tumor response and treating disorders associated with anti-tumor response. The methods generally comprise non-topical administration of purified, isolated quinic acid alkyl acid or a carboxy alkyl ester with a non-toxic carrier or diluent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/504,845 filed Jul. 17, 2009 and allowed Mar. 10, 2011, which, inturn, is a continuation of U.S. patent application Ser. No. 11/270,235filed Nov. 9, 2005 which issued as U.S. Pat. No. 7,579,023 on Aug. 25,2009 which, in turn, is a divisional of U.S. patent application Ser. No.10/093,794 filed Mar. 7, 2002 which issued as U.S. Pat. No. 6,964,784 onNov. 15, 2005, and incorporates its subject matter herein by referencein its entirety. These applications and patents are assigned toOptigenex, Inc.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the isolation, purification andstructural identification of the bioactive component of water extractsof Cat's Claw (Uncaria species). The bioactive component is identifiedas quinic acid lactone. The present invention also is directed to thepharmaceutical use of said bioactive component for enhancing theanti-tumor processes in warm blooded animals.

2. Discussion of the Related Art

Uncaria tomentosa, commonly known as Una de Gato or Cat's Claw, has beenwidely used historically as a natural remedy, and is currently presentin a number of nutritional formulations to treat a large variety ofhealth disorders. To applicant's knowledge, all of the commercialpreparations of Cat's Claw except the water soluble extract (the “Peroextract”) disclosed in U.S. Pat. Nos. 6,039,949, 6,238,675 B1 and6,361,805 (the “Pero patents”) to Pero are based on the oxindolealkaloid content thereof. This is due to Dr. Keplinger's (Austria)discovery, in the early 1960's, of the presence of oxindole alkaloids.(Keplinger, K., Laus, G., Wurm, M., Dierich, M. P., Teppner, H. Uncariatomentosa (WiIld.) DC.—Ethnomedicinal use and new pharmacological,toxicological and botanical results, J. Ethanopharmacology 64:23-34,1999). The Pero extract, the preferred embodiment of which iscommercially available under the name C-MED100®, is a novel Cat's Clawextract quite unlike any other commercial versions in that it containsonly traces of alkaloids (,0.05%). Instead, the Pero extract contains anew class of active ingredients, carboxyl alkyl esters (CAEs), havingdemonstrated efficacy as described and protected in the Pero patents.C-MED-100® is the first product offered in the nutritional industry tosupport both auto-immune and DNA repair enhancing functions, which areof critical importance in reducing the consequences of age-relateddisorders such as autoimmune, inflammatory and neoplastic diseases.References herein to C-MED-100® shall be understood to include the Peroextract, of which C-MED-100® is a preferred embodiment.

The precise chemical identification of the Pero extract's activeingredients has not heretofore been achieved. However, the chemical andbiological characteristics of those ingredients have been sufficientlycompleted to standardize the commercial manufacture of the Pero extract.(See the Pero patents).

C-MED-100®, which is the commercially available Pero extract, isformulated and based on the historical medicinal uses of Cat's Claw, ofwhich an important step is exhaustive hot water extraction forapproximately 18 hours at around 95° C. The extract is thenultrafiltrated to remove high molecular weight (>10,000 MW) toxicconjugates, and spray dried to contain 8-10% carboxy alkyl esters (CAEs)as active ingredients. CAEs were characterized as the only activeingredients of C-MED-100® as a result of their absorption (85%) ontocharcoal. No biological activity was observed in the unabsorbedfraction. Using thin layer chromatography (TLC) as the purificationtool, the active ingredients showed a UV absorption maximum at about 200nm, and reacted with hydroxylamine and ferric chloride, thuscharacterizing them as esters (e.g. CAEs).

Daily oral doses of C-MED-100® between 250-700 mg have provenefficacious in humans. These dosages have been shown to enhanceanti-inflammatory, DNA repair, immuno and anti-tumor processes of warmblooded animals, including humans. (See the Pero patents, Lamm, S.,Sheng, Y., Pero, R. W., Persistent response to pneumococcal vaccine inindividuals supplemented with a novel water soluble extract of Uncariatomentosa, C-Med-100®. Phytomed 8:267-274, 2001; Sheng, Y., Li, L.,Holmgren, K., Pero, R. W., DNA repair enhancement of aqueous extracts ofUncaria tomentosa in a human volunteer study. Phytomed 8: 275-282, 2001;Sheng, Y., Bryngelsson, C., Pero, R. W., Enhanced DNA repair, immunefunction and reduced toxicity of C-MED-100®, a novel aqueous extractfrom Uncaria tomentosa J. of Ethnopharmacology 69:115-126 (2000)).

The CAEs in C-MED-100® are shown to give profound nutritional support asa dietary supplement because the CAEs enhance both DNA repair and immunecell responses, which, in turn, are the critical physiological processesthat regulate aging (See the Pero patents, _(j)Sheng, Y Pero, R. W.,Wagner, H., Treatment of chemotherapy-induced leucopenia in a rat modelwith aqueous extract from Uncaria tomentosa Phytomedicine 7(2): 137-143(2000) and as cited above). Both of these processes involve regulatingthe nuclear transcription kappa beta (NF-kB). NF-kB is well known tocontrol (i) the nuclear events that salvage cells from apoptotic celldeath and (ii) pro-inflammatory cytokine production (Beg, A A andBaltimore, D., An essential role for NF-kB in preventing TNF-α inducedcell death. Science 274: 782-784, 1996; Wang, C-Y, Mayo, M. W., Baldwin,A. S., TNF-α and cancer therapy-induced apoptosis: Potentiation byinhibition of NF-kB. Science 274: 784-787, 1996). Hence, this mechanismdirectly connects induction of apoptosis to programmed cell toxicitywith inhibition of pro-inflammatory cytokine production andinflammation.

Apoptosis is an essential biochemical process in the body that regulatescells from division (replication) into differentiation and toward anincreased functional capacity. Cells entering apoptosis will not only bestimulated to differentiate and increase functionality but willeventually die from this “programmed cell death.” Thus, inducedapoptosis resulting from NF-kB inhibition by C-MED-100® would (i)effectively kill tumor cells, because they would be forced out ofreplication by apoptosis and into eventual death; and simultaneously(ii) increase immune cell responsiveness, because more immune competentcells would be forced to differentiate and would live longer because ofthe parallel enhancement of DNA repair.

NF-kB also sends signals to inflammatory cells instructing them toproduce cytokines (growth factors). These signals, in turn, stimulatephagocytic cells to kill more invading infectious agents, which, atleast in part, is accomplished by producing high levels of oxygen freeradicals. Thus, inhibiting NF-kB has anti-inflammatory propertiesbecause it prevents over-reaction of the inflammatory process that canbe harmful to normal body tissues. In addition, because pro-inflammatorycytokines are a major source of endogenous free radical production inhumans, NF-kB inhibition is antimutagenic by reducing genetic damagethat may accumulate over the years. As fewer radicals are produced,there is less damage to the DNA and less inhibition of natural repair. Aresult is that aging is curtailed.

The Pero extract, preferably C-MED-I00®, is thus an ultimate nutritionalsupplement for anti-aging remedies because it prevents free radicaldamage by NF-kB inhibition, induces differentiation and immune cellresponsiveness by apoptosis, enhances DNA repair, and kills tumor cells,which in turn are the major factors related to aging. (Sheng, Y., Pero,R. W., Amiri, A. and Bryngelsson, C. Induction of apoptosis andinhibition of proliferation and clonogenic growth of human leukemic celllines treated with aqueous extracts of Uncaria tomentosa. AnticancerResearch 18:3363-3368 (1998); Sandoval-Chacon M, Thompson J. H., Zhang XJ, Liu X, Mannick E. E., Sadowicka H., Charbonet R. M., Clark D. A.,Miller M. J. Anti-inflammatory actions of Cat's Claw: the role ofNF-kappa B, Aliment Pharmacol Ther. 12: 1279-1289, 1998; Sandoval M.,Charbonet R. M., Okuhama N. N., Roberts J., Krenova Z., Trentacosti, A.M., Miller M. J. Cat's Claw inhibits TNF-α production and scavenges freeradicals: role in cytoprotection. Free Radicals Biol. Med. 29(1): 71-78,2000). It is beneficial to identify the active component thereof. Byisolating and identifying the active component, it is possible to purifythe component and enhance the pharmaceutical use and increase theefficacy thereof.

The present invention is directed to the isolation, purification andidentification of the CAEs characterized as the active ingredients ofthe Pero extract, which CAES are identified and structurally elucidatedas quinic acid analogs.

BRIEF SUMMARY OF THE INVENTION

If the plant species Uncaria. is hot water extracted, which has been thehistorical practice for medicinal use, and then ultrafiltrated todeplete large molecular weight (>10,000) components, including, forexample, toxic conjugates of tannins, there still remains in thenon-ultrafiltrated fraction, a novel phytomedicinal preparation ofUncaria (e.g. C-MED-100®) having potent immuno, anti-tumor,anti-inflammatory, and DNA repair enhancing properties. In a preferredembodiment of the present invention, C-MED-100® is dissolved in water,spray dried and the spray drying agent (starch) removed by precipitationwith 90% aqueous ethanol. The resultant solution is subjected to thinlayer chromatography (TLC) on silica gel to identify the activeingredient(s) giving the product its efficacy. The 90% ethanolC-MED-100® is spotted on (applied to) TLC plates (silica gel 60 F₂₅₄)and then chromatographed in a system of approximately 1% ammonia ingreater than about 95% ethanol. There is only one area on the TLCchromatogram having biological activity (at R_(f)=0.2-0.3) when elutedwith 1% aqueous ammonia and subsequently bioassayed for the ability tokill tumor cells by induction of apoptosis. The R_(f)=0.2-0.3 compoundshows an ultraviolet absorption maximum in water at about 200 nm,absorbs onto charcoal and is characterized chemically as a CAE byreaction with hydroxylamine and ferric chloride. (Bartos, Colorimetricdetermination of organic compounds by formation of hydroxamic acids,Talanta 27: 583-590, 1980).

In another embodiment of this invention, the biologically active CAEsisolated from the Pero extract, preferably C-MED-100®, are furtherpurified and structurally identified as a quinic acid analog. Elutionfrom silica TLC plates with aqueous ammonia proved to be necessarybecause of very tight binding to silica. Although the R_(f)=0.2-0.3 spotis essentially free from other C-MED-100® components, it containsrelative large amounts of dissolved inorganic silica. In order to removethe inorganic component(s) introduced from the purification scheme onsilica TLC, the 1% aqueous ammonia solution is freeze dried and thenre-dissolved in methanol, leaving behind the solubilized silica. TheR_(f)=0.2-0.3 spot is crystallized from methanol and subsequentlyidentified by chemical analysis as quinic acid.

Thus, one embodiment of the present invention comprises a method forisolating the bioactive component of the Pero extract, preferablyC-MED-100®, comprising: (a). precipitating the spray drying carrier fromthe Pero extract by mixing the extract with distilled water andevaporating the ethanol, and freeze drying the water-dissolved extract;(b) mixing the freeze-dried extract with distilled water and ethanol toobtain a spotting mixture for thin layer chromatography; (c) spottingthe mixture on pre-run TLC plates and chromatographing the plates in asystem of approximately 1% ammonia and ethanol, thereby obtaining afluorescing band with R_(f)=0.2-0.3; (d) scraping off the fluorescingband with R_(f)=0.2-0.3; (e) eluting the scraped band with aqueousammonia and freeze drying the eluted scraped band to dryness to form apowder; (f) extracting the powder with methanol to remove solubilizedsilica gel, leaving a methanol solution; (g) concentrating the methanolsolution; and (h) crystallizing the concentrated solution to obtain thebioactive component.

Another embodiment of the present invention comprises identification ofthe bioactive component of the Pero extract, preferably C-MED-100®,obtained by the foregoing method. In this embodiment the bioactivecomponent exhibits the same properties as the Pero extract and consistsessentially of a quinic acid analog. Preferably, the quinic acid analogis quinic acid lactone.

In another embodiment, the present invention comprises a pharmaceuticalcomposition comprising a pharmaceutically effective amount of thebioactive component of the Pero extract and a nontoxic inert carrier ordiluent. The present invention also includes embodiments which compriseusing the pharmaceutical composition to (i) enhance the immunecompetency of a mammal by inhibiting TNF-α production or inducingapoptosis of white blood cells, comprising administering thepharmaceutical composition in an amount effective to inhibit TNF-αproduction or to induce apoptosis of white blood cells; (ii) treatdisorders associated with the immune system of a mammal by inhibitingTNF-α production or inducing apoptosis of white blood cells, comprisingadministering the pharmaceutical composition in an amount effective toinhibit TNF-α production or to induce apoptosis of white blood cells;(iii) inhibit the inflammatory response of a mammal by inhibiting TNF-αproduction or inducing apoptosis of white blood cells, comprisingadministering the pharmaceutical composition in an amount effective toinhibit TNF-α production or to induce apoptosis of white blood cells;(iv) treat disorders associated with the inflammatory response of amammal by inhibiting TNF-α production or inducing apoptosis of whiteblood cells, comprising administering the pharmaceutical composition inan amount effective to inhibit TNF-α production or to induce apoptosisof white blood cells; (v) enhance the anti-tumor response of a mammal byinducing apoptosis of tumor cells, comprising administering thepharmaceutical composition m an amount effective to induce apoptosis oftumor cells; (vi) treat disorders associated with the response of amammal to tumor formation and growth by inducing apoptosis of tumorcells, comprising administering the pharmaceutical composition in anamount effective to induce apoptosis of tumor cells; and (vii) enhancethe DNA repair processes of a mammal, and, thus, provide anti mutagenicactivity important to treating aging disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the linear regression of UV absorbence versus CAE(estimated as _g/ml using dioctyl phthalate as standard).

DETAILED DESCRIPTION OF THE INVENTION

The method and composition of the present invention are best understoodwith reference to the following examples:

Example 1

Isolation and purification of the bioactive component of the Peroextract. The method of preparation and the composition of the Peroextract, preferably C-MED-100®, are described in the Pero patents whichare incorporated herein by reference. C-MED-100®, a preferred embodimentof the Pero extract is a hot water extraction of Cat's Claw (Uncariatomentosa) carried out for 18-24 hours at 90-100° C. and ultra-filtratedto remove compounds greater than 10,000 molecular weight as previouslydescribed in the Pero patents. C-MED-100® is further prepared for thecommercial market by spray drying the extract with corn starch (NiroF-10 Spray-Drier). Procedures are currently used to purify the activecomponents of C-MED-100® as CAEs and it is understood that theseprocedures would apply to any Pero extract. The procedures are:

1. C-MED-100® work-up for active ingredient estimation: The CAEs inC-MED-100® have very unusual water solubility. They tend to bind totannin and polysaccharide polymers, and so, when dried, are difficult toredissolve in appropriate organic solvents such as ethanol. Thepreferred procedure, and it should be understood that the parametersprovided are approximations and not strict limitations, is:

(a) 100 mg of C-MED-100® is dissolved in 1 ml distilled water in a glasstube for 30 minutes. The dissolved solution is centrifuged at 2000×g for10 minutes. The resulting first supernatant is reserved for analysis.

(b) 200 μl of the first supernatant is placed into a new glass tube, and4.8 ml of 99.7% ethanol is added thereto. The resulting solutioncontains 4 mg/ml C-MED-100® suspended in about 96% ethanol.

(c) The C-MED-100/ethanol solution is vortexed (mixed) and centrifugedat 2000×g to remove insoluble material. The resulting second supernatantis reserved for analysis.

(d) The second supernatant is diluted from a C-MED-100® concentration of4 μg/ml to one of 30-200 μg/ml with 99.7% ethanol for measurement of UVabsorbence. Preferably, concentrations of 60 and 120 μg/ml are examinedas duplicate concentrations for calculation of CAE by UV absorbence.

(e) The UV absorbence at 205 nm for the two concentrations of C-MED-100®(preferably 60 and 120 μg/ml) is measured in a UV spectrophotometer.Because the CAEs in C-MED-100® have a UV maximum absorption at 205 nm,the amount of CAE may be estimated by the degree of UV absorption. Thestandard curve showing the amount of CAE in μg/ml in relation to thedegree of UV absorption is shown in FIG. 1.

(f) Calculation of the concentration of CAEs, in μg/ml is determined bylinear regression analysis of the slope of best fit by the equationy=0.0491x+0.212, where y=UV absorbence values determined andx=concentration of CAE (μg/ml). The two different concentrations ofC-MED-100® (preferably 60 and 120 μg/ml) then serve as the denominatorfor which the calculated CAE from the UV standard curve serves as thenominator in the calculation of percentage CAE in C-MED-100. Inpractice, the two values are averaged.

(g) The foregoing procedure has been validated against a colorimetricprocedure involving conversion of CAE to hydroxamic acids and reactionwith ferric chloride. (Bartos, Colorimetric determination of organiccompounds by formation of hydroxamic acids, Telanta 27: 583-590, 1980).The two procedures give the same estimation of CAE content

2. Analytical procedures for final purification and isolation ofC-MED-100®'s active ingredient. Again, the parameters provided areapproximations and should serve as exemplars not as limitations:

(i) Precipitation of spray drying carrier (corn starch) from crude waterextracts of C-MED-100®: 5 g of C-MED-100® is mixed with 50 ml distilledwater and 950 ml 99.7% ethanol. The ethanol is evaporated off in the airand the resulting solution is freeze dried. Yield is approximately 1 g.

(ii) Silica gel thin layer chromatography (TLC) purification andisolation of C-MED-100®'s active ingredient:

Step 1: To 200 mg C-MED-100® minus the removal of starch (afterprocedure no. 1 above), add 200 μl distilled water and 200 μl 95.5%ethanol. Mix to form a spotting mixture.

Step 2: Spot the spotting mixture of Step I on 4 pre-run TLC plates(Silica gel 60F₂₅₄). The elution system consists of approximately 1% NH₃in at least 95% ethanol. The sole active component is found atR_(f)=0.2-0.3.

Step 3: Scrape off the fluorescing blue band with R_(f)=0.2-0.3. Eluatewith approximately 1% aqueous ammonia and freeze dry to dryness.

Step 4: Extract the powder from Step 3 with methanol to removesolubilized silica gel. Concentrate the methanol solution andcrystallize the active component.

(iii) High pressure liquid chromatography (HPLC) quantitativedetermination of active component: The column preferably is a 3 μm C₁₈column (83 mm.×4.3 mm internal diameter, Perkin Elmer Corp., Norwalk,Conn.). The preferred solvent gradient elution is as follows: Pump Bcontains methanol and pump A contains 1% acetic acid in distilled water.A gradient was run from 10% to 90% over a period of 25 minutes at a flowrate at 1.5 ml/min. Detection is at UV 254 nm. The peak appears at 18minutes into the gradient run.

(iv) Spectrophotometric detection of active ingredients: The activecomponent of C-MED-100® has an absorption maximum in water in the UVrange at about 200 nm. Hence, crude extracts of C-MED-100® also havingan absorption maximum at about 200 nm as well as its purified activecomponents such as CAEs and their corresponding organic acids can beestimated by UV absorption at this wavelength against a known CAEstandard.

An assay of biological activity of C-MED-100®' s active ingredient isprepared as follows: HL-60 W6899 cells are exposed in microculture at5000 cells per well (96-well plates) for 5 days at 37° C. in a CO₂incubator. After incubation, the cells are washed with saline andclonogenicity estimated by MTT assay. Results of the assay aresummarized in Table 1 on page 18 below.

Example 2

Analytical identification of the active ingredient of C-MED-100® asquinic acid. The bioactive component (sample approximately 1 mg)isolated by TLC is completely dissolved in about 0.7 ml D₂0 for NMR withno shift reagent added. The following spectra are recorded:

NMR 020108ta

-   -   1: ¹H    -   2: ¹H/¹H-correlated spectra; COSY    -   3: ¹H/¹³C-correlated spectra; HMBC.    -   4: ¹³C-Dept135.    -   5: ¹H/¹³C-correlated spectra; HMQ.

The ¹H-spectrum contains signals from a main compound. The three¹H-signals at 4.03, 3.90 and 3.43 ppm are found to be signals frommethine-groups (see HMQC). Furthermore, the obtained ¹³C-signals at 66.9B 75.1 correlate to these protons, and theft chemical shifts imply thatthe carbons are bound to oxygen, possibly as CHOH-groups. The threesignals are bound to each other in a straight chain as found in the COSYspectrum.

The main compound also showed ¹H-signals at about 1.72 B 1.99 ppm withcorrelations to ¹³C-signals at about 40 ppm. The HMQC spectrum revealsthat these signals are CH₂-groups and the COSY spectrum implies that theindividual protons in each CH₂-group are unequal.

Judged from the COSY spectrum, the two outer CHOH-groups are bound todifferent CH₂-groups. This gives the following partial structure:

However, as many of the ¹H-¹H-couplings were larger/smaller comparedwith normal couplings it seemed likely that the compound rotation wassterically hindered and therefore a ring system was suggested.Furthermore, as the ¹³C-shifts for the CH₂-groups were near 40 ppm itseemed likely that RI=R2=a carbon atom. This gave the following partialstructure:

No signals that explain X and Y in the compound could be found in theNMR spectra. After the NMR spectra were obtained also MS-analysis wasperformed. The sample was introduced into the MS by infusion. MS spectraon the D₂O solution diluted with acetonitrile (ACN) (50/50) gave themass number of 197 (negative ions, M−D=195). Then the solution wasevaporated by means of a gentle stream of nitrogen and reconstituted inH₂O/ACN (50/50). Here the mass number 192 was achieved (negative ions,M−H=191). In conclusion, the compound mass number is 192 and contains 5exchangeable protons. When combining the information obtained from NMRand MS the following structure is proposed for the main compound:

This structure is quinic acid. Reference spectra obtained usingauthentic quinic acid were identical to that isolated and purified fromC-MED-100®.

Quinic acid, now identified as the active ingredient of C-MED-100®, is aknown compound occurring as an intermediate metabolite in the naturalsynthesis of many aromatic compounds. (Bohm, B A, Shikimic acid(3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid), Chem. Rev. 65:435-466, 1965). Hence, it is disclosed here that quinic acid and itsanalog are expected to occur in many botanical species, giving themadded nutritional and health benefits.

The only known prior art disclosing any medical uses of quinic acid andits analogs is for the treatment of skin wrinkles (U.S. Pat. Nos.5,656,665 and 5,589,505) and of flu as neuroamidase inhibitors (U.S.Pat. Nos. 6,111,132 and 6,225,341). There has been no prior artdisclosure that quinic acid and its analogs might be useful in treatingthe disorders for which C-MED-100® has been useful such as aging,inflammation, immune suppression, and control of tumor growth and DNArepair.

Hence, this disclosure is of these additional uses for quinic acid andits analogs, especially quinic acid lactone. Moreover, quinic acid doesnot give a positive chemical reaction for a CAE. However, upon review ofthis structure, it became apparent that quinic acid might form a quinicacid lactone upon heating, which in turn would react as a CAE. (Fischer,H. O. and Dangschat, G. Hely. Chim Actal 8: 1200, 1935). Furthermore,treating the quinic acid lactone with 1% aqueous ammonia could convertit back to quinic acid. This chemistry was validated using purifiedquinic acid, and establishes that the active ingredient present inC-MED-100® has been synthesized during the historical medicalpreparation of this Cat's Claw product. Example 3 provides thisvalidation

Example 3

This example exploits the biochemical knowledge presented in examples 1and 2 to determine that the active component of C-MED-100® is in factquinic acid lactone. C-MED-100®, quinic acid and quinic acid lactone allabsorb to charcoal, and when they did both the biological activity andUV absorption at 200 nm of C-MED-100® was also removed. This datateaches that the bioactive component of C-MED-100® absorbs maximally at200 nm. The TLC results report that there are only 2 components ofC-MED-100® having such an absorption maxima. The components, located atRf=0.05 and Rf=0.3, when chromatographed in 1% ammonia in ethanol,correspond to quinic acid and quinic acid lactone, respectively.

However, upon evaluation, the bioactive properties of the bioactivecomponent of C-MED-100® could be almost completely accounted for byquinic acid lactone. As a result the anti-aging, anti-inflammatory,immune and DNA repair enhancing and anti-tumor properties of C-MED-100 ®are due to the presence of quinic acid lactone. Those properties arehereby disclosed as attributable to quinic acid lactone.

Table 1 illustrates the relative biochemical activities of (i) theisolated bioactive component of C-MED-100®, (ii) quinic acid, and (iii)quinic acid lactone:

TABLE 1 Comparison of active ingredient of C-MBD-100 ® to quinic acidand its lactone. (Parameters are approximations.) C-MED-100 ® QuinicChemical active Quinic acid Parameter ingredient acid lactone Charcoalabsorption Yes yes yes in water A_(UV) maximum 200 nm 200 nm 200 nm inwater TLC in approximately R_(f) = 0-0.05  R_(f) = 0-0.05 R_(f) =0.2-0.3 1% ammonia in 99% R_(f) = 0.2-0.3 ethanol using A200 nm fordetection Formation of Yes No yes hydroxamic acid/ferric chloride colorcomplex Bioassay efficacy 40 μg/ml >3000 μg/ml 80 μg/ml using IC₅₀ inHL-60 cells Bioassay after 1% >3000 μg/ml >3000 μg/ml >3000 μg/mlaqueous ammonia IC₅₀ HL-60 cells

From the foregoing comparison, it is apparent that the bioactivecomponent in C-MED-100® is, in fact, quinic acid lactone. Specifically,the relative IC₅₀ values for the C-MED-100® bioactive component, quinicacid, and quinic acid lactone confirm that the bioactive componentcannot be quinic acid, per se, but must be an analog thereof, such asquinic acid lactone. The difference in IC₅₀ values for the C-MED-100®bioactive component and quinic acid lactone is not significant, and islikely due to the synergistic effect of other compounds present inC-MED-100®. However, the higher efficacy of the active ingredient,quinic acid lactone, in C-MED-100® than in its pure form indicates thatthe quinic acid lactone is more active in the presence of othernaturally occurring components in C-MED-100® such as quinic acid.

While the invention has been described with respect to certain specificembodiments, it will be appreciated that many modifications and changesmay be made by those skilled in the art without departing from theinvention. It is intended, therefore, by the appended claims to coverall such modifications and changes as may fall within the true spiritand scope of the invention.

1. A method for enhancing the anti-tumor response of a mammal in needthereof by inhibiting TNF-α production or inducing apoptosis of whiteblood cells, comprising administering to said mammal a pharmaceuticalcomposition comprising a pharmaceutically effective amount of apurified, isolated quinic acid alkyl ester having a UV absorptionmaximum at approximately 200 nm and a bioassay efficacy using IC₅₀ inHL-60 cells of less than 100 μg/ml, where said amount is effective toinhibit TNF-α production or to induce apoptosis of white blood cells,and where said administration is other than topical.
 2. A method fortreating disorders associated with the anti-tumor response of a mammalin need thereof by inhibiting TNF-α production or inducing apoptosis ofwhite blood cells, comprising administering to said mammal apharmaceutical composition comprising a pharmaceutically effectiveamount of a purified, isolated quinic acid alkyl ester having a UVabsorption maximum at approximately 200 nm and a bioassay efficacy usingIC₅₀ in HL-60 cells of less than 100 μg/ml, where said amount iseffective to inhibit TNF-α production or to induce apoptosis of whiteblood cells, and where said administration is other than topical.
 3. Amethod for enhancing the anti-tumor response of a mammal in need thereofby inhibiting TNF-α production or inducing apoptosis of white bloodcells, comprising administering to said mammal a pharmaceuticalcomposition comprising a pharmaceutically effective amount of apurified, isolated carboxy alkyl ester where said amount is effective toinhibit TNF-α production or to induce apoptosis of white blood cells,where said carboxy alkyl ester is characterized by having a UVabsorption maximum at approximately 200 nm, absorbs onto charcoal, andreacts with hydroxylamine and ferric chloride, and where saidadministration is other than topical.
 4. A method for treating disordersassociated with the anti-tumor response of a mammal in need thereof byinhibiting TNF-α production or inducing apoptosis of white blood cells,comprising administering to said mammal a pharmaceutical compositioncomprising a pharmaceutically effective amount of a purified, isolatedcarboxy alkyl ester where said amount is effective to inhibit TNF-αproduction or to induce apoptosis of white blood cells, where saidcarboxy alkyl ester is characterized by having a UV absorption maximumat approximately 200 nm, absorbs onto charcoal, and reacts withhydroxylamine and ferric chloride, and where said administration isother than topical.
 5. The method of claim 1 wherein said amount iseffective to inhibit TNF-α production.
 6. The method of claim 1 whereinsaid amount is effective to induce apoptosis of white blood cells. 7.The method of claim 1 wherein said administering includes a nontoxicinert carrier.
 8. The method of claim 1 wherein said administeringincludes a diluent.
 9. The method of claim 2 wherein said amount iseffective to inhibit TNF-α production.
 10. The method of claim 2 whereinsaid amount is effective to induce apoptosis of white blood cells. 11.The method of claim 2 wherein said administering includes a nontoxicinert carrier.
 12. The method of claim 2 wherein said administeringincludes a diluent.
 13. The method of claim 3 wherein said amount iseffective to inhibit TNF-α production.
 14. The method of claim 3 whereinsaid amount is effective to induce apoptosis of white blood cells. 15.The method of claim 3 wherein said administering includes a nontoxicinert carrier.
 16. The method of claim 3 wherein said administeringincludes a diluent.
 17. The method of claim 4 wherein said amount iseffective to inhibit TNF-α production.
 18. The method of claim 4 whereinsaid amount is effective to induce apoptosis of white blood cells. 19.The method of claim 4 wherein said administering includes a nontoxicinert carrier.
 20. The method of claim 4 wherein said administeringincludes a diluent.